Role of serine 214 and tyrosine 171, components of the S2 subsite of alpha-lytic protease, in catalysis.

The function of a hydrogen bond network, comprised of the hydroxyl groups of Tyr 171 and Ser 214, in the hydrophobic S2 subsite of alpha-lytic protease, was investigated by mutagenesis and the kinetics of a substrate analog series. To study the catalytic role of the Tyr 171 and Ser 214 hydroxyl groups, Tyr 171 was converted to phenylalanine (Y171F) and Ser 214 to alanine (S214A). The double mutant (Y171F: S214A) also was generated. The single S214A and double Y171F:S214A mutations cause differential effects on catalysis and proenzyme processing. For S214A, kcat/Km is (4.9 x 10(3))-fold lower than that of wild type and proenzyme processing is blocked. For the double mutant (Y171F:S214A), kcat/Km is 82-fold lower than that of wild type and proenzyme processing occurs. In Y171F, kcat/Km is 34-fold lower than that of wild type, and the proenzyme is processed. The data indicate that Ser 214, although conserved among serine proteases and hydrogen bonded to the catalytic triad [Brayer, G. D., Delbaere, L. T. J., & James, M. N. G. (1979) J. Mol. Biol. 131, 743], is not essential for catalytic function in alpha-lytic protease. A substrate series (in which peptide length is varied) established that the mutations (Y171F and Y171F:S214A) do not alter enzyme-substrate interactions in subsites other than S2. The pH dependence of kcat/Km for Y171F and Y171F:S214A has changed less than 0.5 unit from that of wild type; this suggests the catalytic triad is unperturbed. In wild type, hydrophobic interactions at S2 increase kcat/Km by up to (1.2 x 10(3))-fold with no effect on Km.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase

Proteins have evolved to compensate for detrimental mutations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isomerase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Previous results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a secondsite suppressor.     

Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Delta5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Odelta2...O of Wat504...Tyr-14 Oeta...Tyr-55 Oeta.Tyr-30 Oeta in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26-51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by the additional Y30F/Y55F mutations. The kinetic parameters of the tyrosine mutants with the additional D99N or D99L mutation also support the idea that Asp-99 contributes to catalysis more efficiently in Y14F/Y30F/Y55F than in Y14F. In contrast to the catalytic mechanism of Y14F, the C4 proton of the steroid substrate was found to be transferred to the C6 position in Y14F/Y30F/Y55F with little exchange of the substrate 4beta-proton with a solvent deuterium based on the reaction rate in D2O. Taken together, our findings strongly suggest that the improvement in the catalytic activity of Y14F by the additional Y30F/Y55F mutations is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F.     

Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ⁵-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ⁵-3-ketosteroid to its conjugated Δ⁴-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.     

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.     

Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase

The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base. The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays. TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue. We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.     

15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S(2)) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S(2) values, whereas the S(2) values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.     

Catalytic mechanism of scytalone dehydratase: site-directed mutagenisis, kinetic isotope effects, and alternate substrates.

On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.     

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine.

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH 相似文献   

Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base

3-oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testosteroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through an intermediate dienolate. Residue Asp-38 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6. Both Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) >/= 9.5) function as hydrogen-bond donors to O-3 of the steroid, helping to stabilize the transition states. Mutation of the active-site base Asp-38 to the weakly basic Asn (D38N) has previously been shown to result in a >10(8)-fold decrease of catalytic activity. In this work, we describe the preparation and kinetic analysis of the Ala-38 (D38A) mutant. Unexpectedly, D38A has a catalytic turnover number (k(cat)) that is ca. 10(6)-fold greater than the value for D38N and only about 140-fold less than that for wild type. Kinetic studies as a function of pH show that D38A-catalyzed isomerization involves two groups, with pK(a) values of 4.2 and 10.4, respectively, in the free enzyme, which are assigned to Asp-99 and either Tyr-14 or Tyr-55. A mechanism for D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transition states provided by hydrogen bonding from both Tyr-14 and Tyr-55. This mechanism is supported by the lack of detectable activity of the D38A/D99N, D38A/Y14F, and D38A/Y55F double mutants.     

A conserved tyrosine residue of Saccharomyces cerevisiae leukotriene A4 hydrolase stabilizes the transition state of the peptidase activity

Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining KM and kcat for various amino acid beta-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25-100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization.     

Investigation of the role of a second conserved serine in carboxylesterases via site-directed mutagenesis

Carboxylesterases are enzymes that catalyze the hydrolysis of ester and amide moieties. These enzymes have an active site that is composed of a nucleophile (Ser), a base (His), and an acid (Glu) that is commonly known as a catalytic triad. It has previously been observed that the majority of carboxylesterases and lipases contain a second conserved serine in their active site [Proteins, 34 (1999) 184]. To investigate whether this second serine is also involved in the catalytic mechanism, it was mutated to an alanine, a glycine or a cysteine. Site-directed mutagenesis of this conserved serine resulted in a loss of specific activity, in both the S247G and S247A mutants (5- to 15-fold), which was due to a decrease in the rate of catalysis (kcat). Due to the instability of the S247C mutant no reliable data could be attained. A carbamate inhibitor, carbaryl, was then employed to investigate whether this decrease in the kcat was due to the rate of formation of the acyl-enzyme intermediate (k2) or the rate of deacylation (k3). The S247A mutant was found only to alter k2 (2.5-fold decrease), with no effect on k3. Together with information inferred from a human carboxylesterase crystal structure, it was concluded that this serine provides an important structural support for the spatial orientation of the glutamic acid, stabilizing the catalytic triad so that it can perform the hydrolysis.     

Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr(547) and a water molecule positioned in close proximity to Tyr(547). To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.     

Biochemical characterization and structural analysis of a highly proficient cocaine esterase

The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site.     

Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida

L-Methionine gamma-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of y-family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine y-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in kcat for alpha,gamma-elimination of L-methionine, while the Km remained the same as the wild type enzyme. The Y114F mutant had the reduced kcat by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of kcat for alpha,beta-elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma-elimination process for L-methionine is the rate-limiting determination step in alpha,gamma-elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma-lyase is important in y-elimination of the substrate.     

Modification of a catalytically important residue of indoleglycerol-phosphate synthase from Escherichia coli

The active-site residues of indoleglycerol-phosphate synthase from Escherichia coli were tentatively localized by comparing crystallographic data with the amino acid identities among the known indoleglycerol-phosphate synthase sequences. To test the validity of the resulting model of catalysis one of the residues in the presumptive active site, Lys 55, was changed to serine using oligonucleotide-directed mutagenesis. The specificity constant kcat/Km of the mutant is 3 x 10(4)-times lower than that of the wild-type enzyme, due to a 60-fold decrease in kcat and a 450-fold increase in Km. This finding shows that Lys 55 is important for both catalysis and substrate binding.     

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).     

Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.     

Water in the active site of ketosteroid isomerase

Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.     

Structure and inhibition of the human cell cycle checkpoint kinase, Wee1A kinase: an atypical tyrosine kinase with a key role in CDK1 regulation

Phosphorylation is critical to regulation of the eukaryotic cell cycle. Entry to mitosis is triggered by the cyclin-dependent kinase CDK1 (Cdc2), which is inactivated during the preceding S and G2 phases by phosphorylation of T14 and Y15. Two homologous kinases, Wee1, which phosphorylates Y15, and Myt1, which phosphorylates both T14 and Y15, mediate this inactivation. We have determined the crystal structure of the catalytic domain of human somatic Wee1 (Wee1A) complexed with an active-site inhibitor at 1.8 A resolution. Although Wee1A is functionally a tyrosine kinase, in sequence and structure it most closely resembles serine/threonine kinases such as Chk1 and cAMP kinases. The crystal structure shows that although the catalytic site closely resembles that of other protein kinases, the activation segment contains Wee1-specific features that maintain it in an active conformation and, together with a key substitution in its glycine-rich loop, help determine its substrate specificity.